Synthesis of chiral 2-alkyl amino acids

ABSTRACT

Non-natural amino acids such as 2-alkylated amino acids allow for the synthesis of a wider variety of peptidal and non-peptidal pharmaceutically active agents. A method of preparing a 2-alkyl amino acid involves reacting cysteine (or a salt or an ester thereof) and an aryl carboxylic acid to form a thiazoline ring, stereospecifically alkylating the thiazoline ring, and hydrolyzing the thiazoline ring to obtain a 2-alkylcysteine (or related compound). The present invention also discloses a method of preparing a class of iron chelating agents related to desferrithiocin, all of which contain a thiazoline ring. In this method, an aryl nitrile or imidate is condensed with cysteine, a 2-alkyl cysteine, or a cysteine ester.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.60/381,012, 60/381,021, 60/380,894, 60/380,910, 60/380,880, 60/381,017,60/380,895, 60/380,903, 60/381,013, 60/380,878 and 60/380,909, all ofwhich were filed May 15, 2002. This application also claims the benefitof U.S. Provisional Application No. 60/392,833, filed Jun. 27, 2002. Theentire teachings of the above-referenced applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Alpha-amino acids are useful starting materials in the synthesis ofpeptides, as well as non-peptidal, pharmaceutically activepeptidomimetic agents. In order to enable the synthesis of a largenumber of compounds from an amino acid precursor, it is advantageous tohave naturally occurring and non-naturally occurring amino acids.Non-naturally occurring amino acids typically differ from natural aminoacids by their stereochemistry (e.g., enantiomers), by the addition ofalkyl groups or other functionalities, or both. At this time, theenantiomers of naturally occurring amino acids are much more expensivethan the naturally occurring amino acids. In addition, there are only alimited number of commercially available amino acids that arefunctionalized or alkylated at the alpha-carbon, and often synthesesinvolve the use of pyrophoric or otherwise hazardous reagents. Moreover,the syntheses are often difficult to scale up to a commercially usefulquantity. Consequently, there is a need for new methodologies ofproducing such non-naturally occurring amino acids.

Non-naturally occurring amino acids of interest include the (R)- and(S)-isomers of 2-methylcysteine, which are used in the design ofpharmaceutically active moieties. Several natural products derived fromthese isomers have been discovered in the past few years. These naturalproducts include desferrithiocin, from Streptomyces antibioticus; aswell as tantazole A, mirabazole C, and thiangazole, all from blue-greenalgae. These compounds have diverse biological activities ranging fromiron chelation to murine solid tumor-selective cytotoxicity toinhibition of HIV-1 infection.

Desferrithiocin, deferiprone, and related compounds represent an advancein iron chelation therapy for subjects suffering from iron overloaddiseases. Present therapeutic agents such as desferroxamine requireparenteral administration and have a very short half-life in the body,so that patient compliance and treatment cost are serious problems forsubjects receiving long-term chelation therapy. Desferrithiocin andrelated compounds are effective when orally administered, therebyreducing patient compliance issues. Unfortunately, (S)-2-methylcysteine,which is a precursor to the more active forms of desferrithiocin andrelated compounds, remains a synthetic challenge. Therefore, there is aneed for novel methods of producing 2-methylcysteine at a reasonablecost, and means of isolating the desired enantiomer.

SUMMARY OF THE INVENTION

The present invention includes a method of preparing a compoundrepresented by Structural Formula (I):

or salts thereof; wherein, R₁ is —H or a substituted or unsubstitutedalkyl group; and R₂ is a substituted or unsubstituted alkyl group;comprising the steps of:

-   -   a.) reacting a compound represented by Structural Formula (II):    -    wherein R₃ is —OH, a substituted or unsubstituted alkyloxy        group, or a halogen; with a substituted or unsubstituted aryl        carboxylic acid, thereby forming a substituted thiazoline        represented by Structural Formula (III):    -    wherein Ar is a substituted or unsubstituted aryl group and R₃        is as defined above;    -   b.) reacting the substituted thiazoline with a substituted        oxazolidinone represented by Structural Formula (IV):    -    wherein X is an aryl or an arylalkyl group, thereby forming a        compound represented by Structural Formula (V):    -   c.) alkylating the product of step (b.) with R₂Y, wherein R₂ is        as defined above and Y is a leaving group; thereby forming a        compound represented by Structural Formula (VI):    -    wherein R₂ is as defined above; and    -   d.) hydrolyzing the product of step (c.) (preferably an        inorganic acid such as HCl, HBr or sulfuric acid), thereby        forming the compound represented by Structural Formula (I).

In one embodiment, the present invention is a method of preparing acompound represented by Structural Formula (VII):

or salts thereof; where R₁ is —H or a substituted or unsubstituted alkylgroup; and R₂ is a substituted or unsubstituted alkyl group; comprisingthe steps of:

-   -   a) reacting a compound represented by Structural Formula (VII):    -    wherein R₃ is —OH, a substituted or unsubstituted alkyloxy        group, or a halogen; with a substituted or unsubstituted aryl        carboxylic acid, thereby forming a substituted thiazoline        represented by Structural Formula (IX):    -    wherein Ar is a substituted or unsubstituted aryl group and R₃        is as defined above;    -   b) reacting the substituted thiazoline with a substituted        oxazolidinone represented by Structural Formula (X):    -    wherein X is an aryl or an arylalkyl group, thereby forming a        compound represented by Structural Formula (XI):    -   c) alkylating the product of step (b.) with R₂Y, wherein R₂ is        as defined above and Y is a leaving group; thereby forming a        compound represented by Structural Formula (XII):    -    wherein R₂ is as defined above; and    -   d) hydrolyzing the product of step (c.), thereby forming the        compound represented by Structural Formula (VII).

The present invention also includes method of preparing a compoundrepresented by Structural Formula (XIII):

or salts thereof; where R₁ is —H or a substituted or unsubstituted alkylgroup; and R₂ is a substituted or unsubstituted alkyl group; comprisingthe steps of:

-   -   a) reacting a compound represented by Structural Formula (XIV):    -    wherein R₃ is —OH, a substituted or unsubstituted alkyloxy        group, or a halogen; with a substituted or unsubstituted aryl        carboxylic acid, thereby forming a substituted thiazoline        represented by Structural Formula (XV):    -    wherein Ar is a substituted or unsubstituted aryl group and R₃        is as defined above;    -   b) reacting the substituted thiazoline with a substituted        oxazolidinone represented by Structural Formula (XVI):    -    wherein X is an aryl or an arylalkyl group, thereby forming a        compound represented by Structural Formula (XVII):    -   c) alkylating the product of step (b.) with R₂Y, wherein R₂ is        as defined above and Y is a leaving group; thereby forming a        compound represented by Structural Formula (XVIII):    -    wherein R₂ is as defined above; and    -   d) hydrolyzing the product of step (c.), thereby forming the        compound represented by Structural Formula (XIII).

Preferably, the starting material for the above method is (S)-cysteine,and the product is (S)-2-methylcysteine methyl ester. Alternatively, thestarting material for the above method is (R)-cysteine, and the productis (R)-2-methylcysteine methyl ester. The starting material for theabove method can also be a mixture of (R)- and (S)-cysteine, such as theracemate, and the product is a mixture of (R)- and (S)-2-methylcysteinemethyl ester.

In other embodiments of the invention, the stereochemistry at the4-position of the thiazoline ring (i.e., where the amide is attached)may invert during the alkylation in step (c). This is dependent, forexample, upon the ability of the amide group to exchange position withthe electron pair formed after base deprotonates the thiazoline ring andupon the ability of the 2-oxazolidinone to selectively block thealkylating agent (R₂Y) from approaching a face of the thiazoline andprevent alkylation from occurring on that face. Under circumstances whenstereochemical inversion occurs, to obtain an (S)-alkylated cysteine itmay be advantageous to use (R)-cysteine or a derivative thereof as thestarting material.

In another embodiment, the present invention is a method of preparing acompound represented by Structural Formula (VII):

comprising the steps of:

-   -   a.) reacting a compound represented by Structural Formula (II):    -    wherein R₃ is —OH, a substituted or unsubstituted alkyloxy        group, or a halogen; with a substituted or unsubstituted aryl        carboxylic acid, thereby forming a substituted thiazoline        represented by Structural Formula (III):    -    wherein Ar is a substituted or unsubstituted aryl group and R₃        is as defined above;    -   b.) reacting the substituted thiazoline with a substituted        oxazolidinone represented by Structural Formula (IV):    -    wherein X is an aryl or an arylalkyl group, thereby forming a        compound represented by Structural Formula (V):    -   c.) alkylating the product of step (b.) with R₂Y, wherein R₂ is        as defined above and Y is a leaving group; thereby forming a        compound represented by Structural Formula (VI):    -    wherein R₂ is as defined above; and    -   d.) hydrolyzing the product of step (c.), thereby forming        2-methylcysteine or a salt thereof, and neutralizing the salt,        if present; and    -   e.) coupling (S)-2-methylcysteine with        2,4-dihydroxybenzonitrile, thereby forming the compound        represented by Structural Formula (VII).

Alternative forms of the previous embodiment involve coupling2-hydroxybenzonitrile and (S)-2-methylcysteine or a salt or an esterthereof. Similar syntheses can be conducted with other substitutedbenzonitriles or benzimidates.

Advantages of the present invention include the facile synthesis of a2-alkyl cysteine, or a salt or ester thereof from cysteine, the acidchloride, or an ester thereof. 2-Methylcysteine prepared by the methodof the present invention can be coupled to 2,4-dihydroxybenzonitrile toform 4′-hydroxydesazadesferrithiocin, also referred to as4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid, an iron chelating agent.

DETAILED DESCRIPTION OF THE INVENTION

A useful and efficient method of preparing 2-alkyl cysteine and relatedcompounds involves reacting cysteine (or a related compound, such as acysteine alkyl ester or the acid chloride of cysteine) and an arylcarboxylic acid, to form a thiazoline intermediate. The thiazolineintermediate can be amidated with an oxazolidinone, and thenstereospecifically alkylated. Upon hydrolysis of the thiazoline and theoxazolidinone, a 2-alkyl cysteine (or a related compound) is obtained.

The reaction of cysteine (or a related compound) with an aryl carboxylicacid can occur in a polar solvent, preferably a polar, protic solvent(e.g., methanol, ethanol, water, acetic acid, formic acid, isopropanol,propanol, dimethylformamide, N-ethylacetamide, formaldehyde diethylacetal) with the addition of a base. Acceptable bases include alkalimetal and alkaline earth metal salts, such as sodium hydroxide, sodiummethoxide, sodium ethoxide, sodium carbonate, potassium hydroxide,potassium methoxide, potassium ethoxide, cesium carbonate, calciumcarbonate, potassium carbonate, sodium hexamethyl disilazide, andpotassium hexamethyl disilazide; and trialkylamines such astrimethylamine, triethylamine, and diisopropylethylamine. The arylcarboxylic acid is typically substituted or unsubstituted benzoic acid,but is preferably benzoic acid. The R₃ group of cysteine or a relatedcompound can be a poor leaving group, such that peptide bond formationwith amine-containing compounds is minimized.

The resultant 2-arylthiazoline-4-carboxylic acid can be amidated with a4-aryl-2-oxazolidinone or a 4-arylalkyl-2-oxazolidinone. Preferably, the4-arylalkyl-2-oxazolidinone is 4-benzyl-2-oxazolidinone. The4-aryl-2-oxazolidone can have either an (R) or (S) configuration at the4-position of the oxazolidinone ring, which is selected dependent inpart upon the desired stereochemistry of the alkylated cysteine product.This amidation reaction is typically conducted in a polar solvent (e.g.,acetonitrile, dimethylsulfoxide, dimethylformamide, acetone,hexamethylphosphoramide, methylene chloride, chloroform) in the presenceof a catalytic amount of coupling or promoting agent, such as thionylchloride, dicyclohexylcarbodiimide, diisopropylcarbodiimide,N,N-carbonyldiimidazole, POCl₃, TiCl₄, SO₂ClF, benzotriazol-1-yl diethylphosphate, Ti(O-butyl)₄, N,N,N′,N′-tetramethyl(succinimido)uraniumtetrafluoroborate, 1,1′-carbonylbis(3-methylimidazolium) triflate,Lawesson's reagent, chlorosulfonyl isocyanate, P₂I₄, pyridinium saltswith tributylamine, and a mixture of tributylphosphine andnitrosomethylbenzene.

The 2-aryl-thiazoline-4-carboxamide can be alkylated at the 4-positionof the thiazoline ring by reacting it with base and an alkyating agentof the formula R₂Y, where R₂ and Y are as defined above. The alkylatingagent is typically present in excess, such as about a 2-fold, 3-fold,4-fold, 5-fold, or 10-fold excess. Preferably, R₂ is a substituted orunsubstituted C1-C4 alkyl group. Even more preferably, R₂ is methyl orbenzyl. Y is preferably a halide, such as iodide, chloride, or bromide.Acceptable bases include lithium diisopropylamide (LDA), lithiumdiisopropylamide, lithium N-isopropyl-N-cyclohexylamide, potassiumt-butoxide, sodium t-butoxide, sodium amide, potassium amide, sodiumhydride, and potassium hydride. Titanium(IV) chloride or tin(IV)chloride can also be present in the reaction mixture. When titaniumchloride is present, the reaction is carried out under nitrogen and in awater-free solvent. The reaction temperature is often about −25° C. toabout 0° C. Acceptable solvents are polar, aprotic solvents such asacetone, acetonitrile, dimethylformamide, dioxane, ethyl acetate, ethylether, hexamethylphosphoramide, tetrahydrofuran, and1,2-dimethoxyethane.

After alkylation, the 4-alkyl-2-arylthiazoline-4-carboxyamide isgenerally hydrolyzed. Typically, hydrolysis involves reacting the4-alkyl-2-arylthiazoline-4-carboxamide with an appropriate amount ofbase in a polar, protic solvent. Preferred bases include alkali andalkaline earth metal salts such as lithium hydroxide, potassiumcarbonate, calcium carbonate, and cesium carbonate. Preferred solventsinclude C1-C4 alcohols such as methanol, ethanol, propanol, isopropanol,butanol, isobutanol, and t-butanol. Methanol is an especially preferredsolvent. In this reaction, the solvent is also a reactant, such thatwhen an alcohol of formula R₁OH is the solvent, R₁O becomes part of thenewly-formed ester. For example, when methanol is the solvent, theproduct is a 2-alkylcysteine methyl ester. The ester moiety can behydrolyzed by reacting the cysteine ester with a sufficient quantity ofacid or base to remove the methoxy group. The acid or base used forhydrolysis preferably does not react with or cleave, except to form asalt, other moieties of the 2-alkylcysteine.

Chiral amino acid products, either enantiomers or diastereomers, of theabove syntheses can be purified by an additional resolving step.Typically, amino acids are resolved by forming a diastereomeric saltwith an amino acid and a chiral amine. Suitable chiral amines includearylalkylamines such as (R)-1-phenylethylamine, (S)-1-phenylethylamine,(R)-1-tolylethylamine, (S)-1-tolylethylamine, (R)-1-phenylpropylamine,(S)-1-propylamine, (R)-1-tolylpropylamine, and (S)-1-tolylpropylamine.Resolution of chiral compounds using diastereomeric salts is furtherdescribed in CRC Handbook of Optical Resolutions via Diastereomeric SaltFormation by David Kozma (CRC Press, 2001), which is incorporated hereinby reference in its entirety.

Alternatively, 2-alkyl amino acids and functionalized derivativesthereof (e.g., esters) can be purified by emulsion crystallization, asdescribed in U.S. Pat. Nos. 5,872,259, 6,383,233 and 6,428,583, whichare incorporated herein by reference. Briefly, emulsion crystallizationis a process for separating a desired substance from an aggregatemixture. The process involves forming a three phase system, the firstphase comprising the aggregate mixture, the second phase being liquidand comprising a transport phase, and the third phase comprising asurface upon which the desired substance can crystallize. A chemicalpotential exists for crystal growth of the desired substance in thethird phase of the system, thereby creating a flow of the desiredsubstance from the first phase through the second phase to the thirdphase, where the desired substance crystallizes and whereby anequilibrium of the activities of the remaining substances in theaggregate mixture is maintained between the first phase and the secondphase.

In one example of emulsion crystallization, a solution of the racemicmixture is supersaturated (by either cooling, adding a solvent in whichone or more components are sparingly soluble or by evaporation of thesolution). Ultrasonication eventually helps the process of forming anemulsion. The mixture is then seeded with crystals of the desired,optically active acid along with an additional quantity of surfactantand an anti-foaming agent. The desired product usually crystallizes outand can be separated by filtration. Further details of emulsioncrystallization for an amino acid derivative can be found in Example 2.

Once the 2-alkyl amino acids or functionalized derivatives have beenresolved, the desired isomer can be isolated. Typically, a (S)-2-aminoacid, a salt, or an ester thereof is isolated. Preferably,(S)-2-methylcysteine or (S)-2-methylcysteine methyl ester is isolated.

Cysteine, a 2-alkylcysteine such as (S)-2-methylcysteine, or a cysteinealkyl ester can be coupled to a substituted or unsubstituted arylnitrile such as a substituted or unsubstituted benzonitrile. Preferably,the substituents on benzonitrile will not interfere with the couplingreaction. In a preferred embodiment, (S)-2-methylcysteine is coupled to2,4-dihydroxybenzonitrile to form4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid (also known as 4′-hydroxydesazadesferrithiocin).

Typically, coupling of cysteine, a 2-alkylcysteine, or a cysteine alkylester and a substituted or unsubstituted benzonitrile includesconverting the benzonitrile into a benzimidate. The benzimidate can beformed, for example, by reacting the benzonitrile with an alcohol suchas methanol, ethanol, n-propanol, or isopropanol in the presence of anacid such as hydrochloric acid. Alternatively, cysteine or a relatedcompound can be coupled directly with a benzimidate. The benzimidate isthen reacted with the cysteine (or related compound) under basicconditions. Acceptable bases include trimethylamine, triethylamine,triphenylamine, dimethylamine, diethylamine, diphenylamine,diisopropylamine, diisopropylethylamine, 1,4-diazabicyclo[2.2.2]octane(DABCO), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), and the like. Thereaction between the benzimidate and the cysteine results in thethiazoline (or 4,5-dihydrothiazole) containing product. When forming thebenzimidate from a hydroxylated benzonitrile (e.g.,2,4-dihydroxybenzonitrile), the hydroxyl groups are advantageouslyprotected (e.g., with a substituted or unsubstituted alkyl or arylalkylgroup such as a benzyl group). The protecting groups are subsequentlycleaved, typically by catalytic hydrogenation.

The methods of the claimed invention can be used to manufacture otherrelated desferrithiocin analogs and derivatives. Examples of suchanalogs include those described in U.S. Pat. Nos. 5,840,739, 6,083,966,6,159,983, 6,521,652 and 6,525,080 to Raymond J. Bergeron, Jr., thecontents of which are incorporated herein by reference. Additionalexamples can be found in PCT/US93/10936, PCT/US97/04666, andPCT/US99/19691, the contents of which are incorporated by reference.

Suitable benzonitriles and benzimidates for use in the above couplingreaction can be synthesized by methods described in U.S. applicationSer. Nos. 60/381,013, 60/380,878 and 60/380,909, all filed May 15, 2002,the entire teachings of which are incorporated herein by reference.

An alkyl group is a hydrocarbon in a molecule that is bonded to oneother group in the molecule through a single covalent bond from one ofits carbon atoms. Alkyl groups can be cyclic or acyclic, branched orunbranched, and saturated or unsaturated. Typically, an alkyl group hasone to about 24 carbons atoms, or one to about 12 carbon atoms. Loweralkyl groups have one to four carbon atoms and include methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl and tert-butyl.

Aromatic or aryl groups include carbocyclic aromatic groups such asphenyl, p-tolyl, 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl.Aryl groups also include heteroaryl groups include N-imidazolyl,2-imidazole, 2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyranyl, 3-pyranyl,3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl and 5-oxazolyl.

Aryl groups also include fused polycyclic aromatic ring systems in whicha carbocyclic, alicyclic, or aromatic ring or heteroaryl ring is fusedto one or more other heteroaryl or aryl rings. Examples include2-benzothienyl, 3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl,2-indolyl, 3-indolyl, 2-quinolinyl, 3-quinolinyl, 2-benzothiazole,2-benzooxazole, 2-benzimidazole, 2-quinolinyl, 3-quinolinyl,1-isoquinolinyl, 3-quinolinyl, 1-isoindolyl and 3-isoindolyl.

Suitable substituents for alkyl groups include —OH, halogen (—Br, —Cl,—I and —F), —O(R′), —O—CO—(R′), —CN, —NO₂, —COOH, ═O, —NH₂, —NH(R′),—N(R′)₂, —COO(R′), —CONH₂, —CONH(R′), —CON(R′)₂, —SH, —S(R′), andguanidine. Each R′ is independently an alkyl group or an aryl group.Alkyl groups can additionally be substituted by a aryl group (e.g. analkyl group can be substituted with an aromatic group to form anarylalkyl group). A substituted alkyl group can have more than onesubstituent.

Suitable substituents for aryl groups include —OH, halogen (—Br, —Cl, —Iand —F), —O(R′), —O—CO—(R′), —CN, —NO₂, —COOH, ═O, —NH₂, —NH(R′),—N(R′)₂, —COO(R′), —CONH₂, —CONH(R′), —CON(R′)₂, —SH, —S(R′), andguanidine. Each R′ is independently an alkyl group or an aryl group.Aryl groups can additionally be substituted by an alkyl orcycloaliphatic group (e.g. an aryl group can be substituted with analkyl group to form an alkylaryl group such as tolyl). A substitutedaryl group can have more than one substituent.

Leaving groups are typically weak bases. Suitable leaving groups includehalogen, tosyl, triflyl, brosyl, p-nitrophenyl, 2,4-dinitrophenyl, andmesyl groups. Halogens include bromine, chlorine, and iodine.

Also included in the present invention are salts of the disclosed2-alkylcysteines. For example, cysteines can also be present in theanionic, or conjugate base, form, in combination with a cation. Suitablecations include alkali metal ions, such as sodium and potassium ions,alkaline earth ions, such as calcium and magnesium ions, andunsubstituted and substituted (primary, secondary, tertiary andquaternary) ammonium ions. Suitable cations also include transitionmetal ions such as manganese, copper, nickel, iron, cobalt, and zinc.Basic groups such as amines can also be protonated with a counter anion,such as hydroxide, halogens (chloride, bromide, and iodide), acetate,formate, citrate, ascorbate, sulfate or phosphate.

EXAMPLE 1

(S)-Cysteine is reacted with benzoic acid to form2-phenylthiazoline-4-carboxylic acid. 2-Phenylthiazoline-4-carboxylicacid is amidated with 4-benzyloxazolidone. The amidated2-phenylthiazoline-4-carboxylic acid is alkylated with methyl iodide inthe presence of TiCl₄ and lithium diisopropylamide. The alkylatedspecies is hydrolyzed by lithium hydroxide in methanol to obtain(S)-2-methylcysteine methyl ester.

EXAMPLE 2

All compounds were used without further purification. The surfactantsRhodafac RE 610 and Soprophor FL were obtained from Rhône-Poulenc,Surfynol 465 from Air Products, Synperonic NP 10 from ICI and sodiumlauryl sulfate from Fluka. For agitation a shaking machine was used(Buhler KL Tuttlingen). Purities of the resulting crystals were measuredby using a PolarMonitor polarimeter (IBZ Hannover). Ethanol was used asthe solvent. The total crystal quantity was dissolved in a 1 mL cell at20° C.)

45 mg of (R,R)- and (S,S)-amino acid derivatives were dissolved in 1 mlof a mixture of 20% v/v 2-hexanol, 12% v/v Rhodafac RE 610, 6% v/vSoprophor FL and 62% v/v water by heating to 80° C. in a 5 mL vial.After the organic derivative was completely dissolved the microemulsionwas cooled down to room temperature and agitated using a shaking machine(420 rpm). During two hours no spontaneous crystallization was observed.The mixture was then seeded with two drops of a dilute, finely groundsuspension of pure (S,S)-(−) amino acid or its ester crystals grownunder similar conditions. After 2 hours of agitation the resultingcrystals were filtered off, washed with water and dried in a gentlenitrogen stream.

EXAMPLE 3

35 mg of R- andS-4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4-carboxylic acidwere dissolved in 1 ml of a mixture of 9% N-methyl-pyrrolidone, 9% v/v2-hexanol, 10% v/v Rhodafac RE 610, 5% v/v Soprophor FL and 68% v/vwater by heating to 50° C. in a 5 mL vial. After the product wascompletely dissolved, the microemulsion was cooled down to roomtemperature and agitated with a shaking machine (350 rpm). During twohours, no spontaneous crystallisation was observed. The mixture was thenseeded with two drops of a dilute, finely ground suspension of pureS-product crystals grown under similar conditions. After two hours ofshaking, the resulting crystals were filtered off, washed with water anddried in a gentle nitrogen stream. The procedure yielded 5.4 mg (15.4%)of colorless crystals, with a greater than 90% purity of the Sentantiomer.

EXAMPLE 4

4.00 g (S)-2-methylcysteine hydrochloride (23.3 mmol, 1.0 meq) and 3.14g 2,4-dihydroxy benzonitrile (23.3 mmol, 1.0 meq) were suspended in 40mL ethanol. After degassing this mixture with nitrogen (30 min) 4.95 gtriethylamine (6.8 mL, 48.9 mmol, 2.05 meq) were added. The obtainedsuspension was heated under reflux in an atmosphere of nitrogen for 20hours and then cooled to room temperature. From this suspension ethanolwas evaporated under reduced pressure until an oil (20% of the initialvolume) was obtained. This oil was dissolved in 50 mL water. Thesolution was adjusted to pH 7.5 with 1.20 ml 20% KOH and was extractedtwo times each with 20 mL methyl t-butyl ether (MTBE). The aqueous layerwas separated, adjusted with 20% KOH to pH 11 and again extracted twotimes each with 20 mL MTBE. After separating the aqueous layer the pHwas set with concentrated HCl to 7.5 and traces of MTBE were distilledoff. Then the aqueous solution was acidified with 1.50 ml concentratedHCl to pH 1.5. The product precipitated. This suspension was stirred at4° C. for 1 hour. Then the precipitate was filtered, washed two timeseach with 10 mL water (5° C.) and dried at 45° C. under vacuum. Thereaction yielded 5.17 g (87.6%) of crude4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylicacid product. ¹H-NMR showed no significant impurity.

EXAMPLE 5

2,4-Dibenzyloxybenzonitrile (0.121 mol) was dissolved in 5.85 g (0.127mol) ethanol and 19.4 ml 1,2-dimethoxyethane in a double walled reactor.This solution was cooled to −5° C., stirred and saturated with dry HClgas over 5 hours at 0-3° C. The reaction mixture was stirred overnightat 2-4° C. under nitrogen. During this time, a product crystallized. Thewhite crystals were filtered off, washed with 1,2-dimethoxyethane (5°C., three times each with 13 ml) and dried. A total of 30 of theprotected ethyl benzimidate was isolated (Yield 88.4%, purity 98.9%).

The protected ethyl benzimidate described above was dissolved inmethanol to generate a 10% solution and was catalytically hydrogenatedat room temperature using 5% Pd/C as a catalyst. The reaction wascompleted after 8 hours. The solution was filtered and the solventevaporated to yield the deprotected product as an orange-yellow solid.The reaction yielded 19.6 g (94%) of product.

In contrast, the formation of the imidate with 2,4 dihydroxybenzonitrilewas a low yielding process, generating the desired product in only 20%yield and with less than desired purity. While this invention has beenparticularly shown and described with references to preferredembodiments thereof, it will be understood by those skilled in the artthat various changes in form and details may be made therein withoutdeparting from the scope of the invention encompassed by the appendedclaims.

1. A method of preparing a compound represented by Structural Formula(I):

or salts thereof; wherein, R₁ is —H or a substituted or unsubstitutedalkyl group; and R₂ is a substituted or unsubstituted alkyl group;comprising the steps of: a) reacting a compound represented byStructural Formula (II):

 wherein R₃ is —OH, a substituted or unsubstituted alkyloxy group, or ahalogen; with a substituted or unsubstituted aryl carboxylic acid,thereby forming a substituted thiazoline represented by StructuralFormula (III):

 wherein Ar is a substituted or unsubstituted aryl group and R₃ is asdefined above; b) reacting the substituted thiazoline with a substitutedoxazolidinone represented by Structural Formula (IV):

 wherein X is an aryl or an arylalkyl group, thereby forming a compoundrepresented by Structural Formula (V):

c) alkylating the product of step (b) with R₂Y, wherein R₂ is as definedabove and Y is a leaving group; thereby forming a compound representedby Structural Formula (VI):

 wherein R₂ is as defined above; and d) hydrolyzing the product of step(c), thereby forming the compound represented by Structural Formula (I).2. The method of claim 1, wherein Ar is a substituted or unsubstitutedphenyl group.
 3. The method of claim 2, wherein R₂ is a C1-C4 alkylgroup.
 4. The method of claim 3, wherein R₁ is a C1-C4 alkyl group. 5.The method of claim 4, wherein step (d) comprises reacting the productof step (c) with base and R₁OH, wherein R₁ is as defined above.
 6. Themethod of claim 5, wherein Ar is phenyl.
 7. The method of claim 6,wherein X is benzyl.
 8. The method of claim 7, wherein R₂ is methyl. 9.The method of claim 8, wherein R₁ is methyl.
 10. The method of claim 9,wherein R₃ is —OH.
 11. The method of claim 10, wherein the enantiomersof the product of step (d) are resolved.
 12. The method of claim 11,wherein (S)-2-methylcysteine is isolated from the enantiomers.
 13. Amethod of preparing a compound represented by Structural Formula (VII):

or salts thereof; wherein, R₁ is —H or a substituted or unsubstitutedalkyl group; and R₂ is a substituted or unsubstituted alkyl group;comprising the steps of: a) reacting a compound represented byStructural Formula (VII):

 wherein R₃ is —OH, a substituted or unsubstituted alkyloxy group, or ahalogen; with a substituted or unsubstituted aryl carboxylic acid,thereby forming a substituted thiazoline represented by StructuralFormula (IX):

 wherein Ar is a substituted or unsubstituted aryl group and R₃ is asdefined above; b) reacting the substituted thiazoline with a substitutedoxazolidinone represented by Structural Formula (X):

 wherein X is an aryl or an arylalkyl group, thereby forming a compoundrepresented by Structural Formula (XI):

c) alkylating the product of step (b) with R₂Y, wherein R₂ is as definedabove and Y is a leaving group; thereby forming a compound representedby Structural Formula (XII):

 wherein R₂ is as defined above; and d) hydrolyzing the product of step(c), thereby forming the compound represented by Structural Formula(VII).
 14. The method of claim 13, wherein Ar is a substituted orunsubstituted phenyl group.
 15. The method of claim 14, wherein R₂ is aC1-C4 alkyl group.
 16. The method of claim 15, wherein R₁ is a C1-C4alkyl group.
 17. The method of claim 16, wherein step (d) comprisesreacting the product of step (c) with base and R₁OH, wherein R₁ is asdefined above.
 18. The method of claim 17, wherein Ar is phenyl.
 19. Themethod of claim 18, wherein X is benzyl.
 20. The method of claim 19,wherein R₂ is methyl.
 21. The method of claim 20, wherein R₁ is methyl.22. The method of claim 21, wherein R₃ is —OH.
 23. A method of preparinga compound represented by Structural Formula (XIII):

or salts thereof; wherein, R₁ is —H or a substituted or unsubstitutedalkyl group; and R₂ is a substituted or unsubstituted alkyl group;comprising the steps of: a) reacting a compound represented byStructural Formula (XIV):

 wherein R₃ is —OH, a substituted or unsubstituted alkyloxy group, or ahalogen; with a substituted or unsubstituted aryl carboxylic acid,thereby forming a substituted thiazoline represented by StructuralFormula (XV):

 wherein Ar is a substituted or unsubstituted aryl group and R₃ is asdefined above; b) reacting the substituted thiazoline with a substitutedoxazolidinone represented by Structural Formula (XVI):

 wherein X is an aryl or an arylalkyl group, thereby forming a compoundrepresented by Structural Formula (XVII):

c) alkylating the product of step (b) with R₂Y, wherein R₂ is as definedabove and Y is a leaving group; thereby forming a compound representedby Structural Formula (XVIII):

 wherein R₂ is as defined above; and d) hydrolyzing the product of step(c), thereby forming the compound represented by Structural Formula(XIII).
 24. The method of claim 23, wherein Ar is a substituted orunsubstituted phenyl group.
 25. The method of claim 24, wherein R₂ is aC1-C4 alkyl group.
 26. The method of claim 25, wherein R₁ is a C1-C4alkyl group.
 27. The method of claim 26, wherein step (d) comprisesreacting the product of step (c) with base and R₁OH, wherein R₁ is asdefined above.
 28. The method of claim 27, wherein Ar is phenyl.
 29. Themethod of claim 28, wherein X is benzyl.
 30. The method of claim 29,wherein R₂ is methyl.
 31. The method of claim 30, wherein R₁ is methyl.32. The method of claim 31, wherein R₃ is —OH.
 33. A method of preparinga compound represented by Structural Formula (VII):

comprising the steps of: a) reacting a compound represented byStructural Formula (II):

 wherein R₃ is —OH, a substituted or unsubstituted alkyloxy group, or ahalogen; with a substituted or unsubstituted aryl carboxylic acid,thereby forming a substituted thiazoline represented by StructuralFormula (III):

 wherein Ar is a substituted or unsubstituted aryl group and R₃ is asdefined above; b) reacting the substituted thiazoline with a substitutedoxazolidinone represented by Structural Formula (IV):

 wherein X is an aryl or arylalkyl group, thereby forming a compoundrepresented by Structural Formula (V):

c) alkylating the product of step (b) with R₂Y, wherein R₂ is as definedabove and Y is a leaving group; thereby forming a compound representedby Structural Formula (VI):

 wherein R₂ is as defined above; and d) hydrolyzing the product of step(c), thereby forming (S)-2-methylcysteine or a salt thereof, andneutralizing the salt, if present; e) coupling (S)-2-methylcysteine with2,4-dihydroxybenzonitrile, thereby forming the compound represented byStructural Formula (VII).